Combining Chemical Reaction with Single Cell Mass Spectrometry for Real-time Quantification of Nitric Oxide (NO) Inside Live Single Cells

在化学系化学测量与成像（CMI）项目和促进竞争性研究（EPSCoR）项目的支持下，俄克拉何马大学的杨志波和他的团队研究了单细胞中一氧化氮（NO）的可能检测和定量方法。一氧化氮是一种对人类健康和疾病至关重要的小分子。一氧化氮的产生和浓度受到严格调控，因为它是健康生物学中重要的信号分子；NO浓度的偏差也可能导致生物功能障碍；因此，准确检测一氧化氮水平的方法，特别是在生命系统中，需求量很大。由于细胞内NO的浓度因细胞而异，因此需要在单细胞水平上进行有意义的研究。然而，检测和定量单个细胞中的NO是非常具有挑战性的，主要是因为它的不稳定性和低丰度（例如，细胞直径约为人类头发直径的1/10）。杨博士和他的团队将设计一种微型设备，可以与灵敏的分析工具质谱（MS）耦合。该装置可以直接从单个细胞中提取NO，然后通过在线化学反应将其转化为稳定的分子，用于ms的灵敏检测和准确定量，这项新技术可能为单水平NO等氧化剂的测量提供新的分析工具。该暑期拓展计划预计将通过学校-大学-社区合作为俄克拉荷马州高中的科学教师提供课程发展。推广项目的产品（例如，讲座材料、课程和调查结果）将被其他高中和普通公众使用。此外，进行研究将为本科生和研究生提供专业发展。NO是一种小的生物活性分子，在许多细胞功能中发挥重要作用，这些功能与神经元信号传导、免疫反应和人类疾病有关。一氧化氮的功能与其在细胞中的丰度有关。由于几乎在所有生物系统中都有报道的细胞异质性，细胞间NO的丰度显著不同。单个细胞中NO的定量可以大大提高我们对NO在生物系统中的功能和机制的理解。然而，这些研究是非常具有挑战性的，主要是因为单细胞的极小尺寸和NO的反应性，弥漫性。本研究将化学反应与单细胞质谱（SCMS）相结合，以检测和量化单细胞中的NO。细胞系将被用作产生内源性和外源性NO的模型系统。建立的单探针SCMS实验装置将与离线化学反应相结合用于NO测量。关键的诊断反应涉及氨氯地平（AML）的双电子氧化到脱氢氨氯地平（DAM）。由于该反应涉及简单地从AML中去除“H-H”元素，因此DAM的观察是NO的间接测量，并且对于控制其他原则上可能从AML中产生DAM的双电子氧化反应将是重要的。也许最值得注意的是，作为这些研究的一部分，一种新的设备，细长单探针（essingle -probe）正在开发中，并将用于实时反应（rrSCMS）分析单细胞中的NO。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry (CHE) and the Established Program to Stimulate Competitive Research (EPSCoR), Zhibo Yang and his group at the University of Oklahoma are investigated methods for the possible detection and quantification of nitric oxide (NO) in single cells. NO is a small molecule important for human health and diseases. The production and concentration of NO is tightly regulated as this is an important signaling molecule in healthy biology; deviations in NO concentration can also potentially lead to biological dysfunction; hence methods to accurately detect NO levels, particularly in living systems are in high demand. Because the concentrations of NO in cells are very different from cell to cell, meaningful studies need to be performed at the single-cell level. However, detecting and quantifying NO in single cells is very challenging, primarily because of its instability and low abundance (e.g., a cell diameter is ~1/10 of that of human hair). Dr. Yang and his group will design a microscale device that can be coupled to a sensitive analytical tool, mass spectrometry (MS). This device can directly extract NO from single cells, and then use online chemical reactions to convert it into a stable molecule for sensitive detection and accurate quantification using MS. This new technique can potentially offer a new analytical tool for the measurement of oxidants such as NO at the single level. The summer outreach program is expected to provide lesson development for science teachers at Oklahoma high schools through school-university-community collaborations. The products (e.g., lecture materials, lessons, and survey results) from the outreach program will be accessible by other high schools and general public. In addition, conducting the research will provide professional development for undergraduate and graduate students.NO is a small bioactive molecule playing important roles in numerous cell functions that are relevant to neuronal signaling, immune response, and human disease. The functions of NO are related to its abundance in cells. Due to cell heterogeneity, which has been reported in nearly all biological systems, the abundance of NO significantly varies from cell to cell. Quantification of NO in individual cells could substantially improve our understanding of the functions and mechanisms of NO in biological systems. However, these studies are very challenging, primarily because of the extremely small size of single cells and the reactive, diffusive nature of NO. This proposal combines chemical reactions with single cell mass spectrometry (SCMS) to detect and quantify NO in single cells. Cell lines will be used as model systems to produce endogenous and exogenous NO. An established single-probe SCMS experimental setup will be combined with off-line chemical reactions for NO measurement. The key diagnostic reaction involves the two-electron oxidation of amlodipine (AML) to dehydroamlodipine (DAM ). Since this reaction involves the simple removal of the elements of "H-H" from AML, the observation of DAM is an indirect measure of NO, and it will be important to control for other two-electron oxidation reactions that could, in principle, produce DAM from AML. Perhaps most notably, as part of these studies, a new device, the elongated single-probe (eSingle-probe), is being developed and will be used for real-time reactive (rrSCMS) analysis for NO in single cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.